Arrangements and methods for facilitating photoluminescence imaging

ABSTRACT

Exemplary systems and methods for obtaining a photoluminescence radiation from at least one portion of a sample can be provided. For example, using the exemplary embodiment, it is possible to receive a first radiation and disperse the first radiation into at least one second radiation and at least one third radiation. The second and third radiations can be provided to different locations of the portion. In addition, it is possible to receive the photoluminescence radiation from the portion based on the second and third radiations.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is based upon and claims the benefit of priority fromU.S. Patent Application Ser. No. 60/727,215, filed Oct. 14, 2005, theentire disclosure of which is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under Grant No.FA9550-04-1-0079 awarded by U.S. Department of the Air Force and GrantNo. AR007098 awarded by the Public Health Services/National Institutesof Health. The Government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention generally relates to arrangements and methods forfacilitating photoluminescence imaging, and particularly for, e.g.,obtaining fluorescence images via endoscopes, catheters, orsmall-diameter probes.

BACKGROUND OF THE INVENTION

In vivo fluorescence macro- and microscopic imaging is increasinglybeing used for clinical disease diagnosis and small animal research. Inorder to extend fluorescence imaging for a wide range of basic andclinical applications, it may be preferable to utilize flexible,miniaturized endoscopes. The performance of high quality fluorescentimaging procedures through a miniature flexible probe may be difficultdue to the inability to incorporate a rapid beam scanning mechanism atthe distal end of miniature probes and the limited number of opticalfibers that can fit within the confines of small diameter fiber-opticimaging bundles.

Conventional procedures which apparently implemented fluorescenceimaging through probes with a diameter of less than 2 mm have beenperformed using fiber optic bundles. For example, probes which vary indiameter from 600 μm to 1.8 mm have been used to obtain images ofvessels in the mouse cremaster muscle, and which visualized labeledcirculating cells. (See E. Laemmel et al., “Fibered confocalfluorescence microscopy (Cell-viZio™) facilitates extended imaging inthe field of microcirculation—A comparison with intravital microscopy,”J. Vasc. Res., Vol. 41(5), 400 (2004)). As described in thispublication, images of cells labeled with Fluorescein Isothiocyanate(“FITC”) (e.g., excitation with 488 nm) were obtained at 12 Hz with amaximal field of view of 400 μm×280 μm through probes with ˜10,000optical fibers.

An 800 μm diameter endoscope with 10,000 optical fibers which can beused with Cy5.5 and Cy7, excited at 673 nm can also be utilized. (See M.A. Funovics et al., “Miniaturized multichannel near infrared endoscopefor mouse imaging,” Molecular Imaging, Vol. 2(4), 350 (2003)). Theimaging tip, which has a 56° field of view in water, can also facilitatewhite light reflectance imaging with a resolution of 7 line pairs permillimeter, as determined with an USAF 1951 resolution target. Exemplaryimages were presented from mouse vasculature and of protease activity inan ovarian tumor with rates ranging from 3 to 10 Hz. (See M. A. Funovicset al., “Catheter-based in vivo imaging of enzyme activity and geneexpression: Feasibility study in mice,” Radiology, Vol. 231(3), 659(2004)). According to this publication, tumors expressing greenfluorescent protein were also observed.

Spectral encoding has been previously demonstrated for reflectanceimaging. (See G. J. Tearney et al., “Spectrally encoded confocalmicroscopy,” Opt. Lett., Vol. 23(15), 1152 (1998); and G. J. Tearney etal., “Spectrally encoded miniature endoscopy,” Optics Letters, Vol.27(6), 412 (2002)). In this exemplary technique, broadband light from anoptical fiber may be dispersed by a grating, and focused onto a line onthe sample. In this matter, the image does not have to be scanned inthis dimension. A reflected light returns through the lens, grating, andoptical fiber and the spectrally encoded image is then decoded viaheterodyne Fourier transform spectroscopy (see G. J. Tearney et al.,“Spectrally encoded confocal microscopy,” Opt. Lett., Vol. 23(15), 1152(1998)) or with another grating in conjunction with a CCD detector (seeG. J. Tearney et al., “Spectrally encoded miniature endoscopy,” OpticsLetters, Vol. 27(6), 412 (2002)).

The transverse dimension can then be scanned by, for example, rotatingthe fiber and distal optics, which can be implemented in small diameterprobes. (See G. J. Tearney et al., “Scanning single-mode fiber opticcatheter-endoscope for optical coherence tomography,” Opt. Lett., Vol.21(7), 543 (1996)). Using this conventional technique, the number ofresolvable points (n) along one spectrally encoded line can bedetermined by the spectral bandwidth (Δλ), center wavelength (λ₀), beamdiameter (d), and grating:

$\begin{matrix}{{n \cong \frac{{\Delta\lambda}\;{dG}}{\lambda_{0}{\cos\left( \theta_{i} \right)}}},} & (1)\end{matrix}$where G and θ_(i) are the grating groove density and incidence angle,respectively. (See G. J. Tearney et al., “Spectrally encoded miniatureendoscopy,” Optics Letters, Vol. 27(6), 412 (2002)).

The spectrally encoded photoluminescient techniques are generally basedon a similar concept. In this exemplary embodiment, the fluorescenceemission may be Stokes shifted, and the spatial locations are generallyno longer uniquely related to the detected wavelengths. As a result,spectroscopic methods and arrangements implementing the same may not beeffective for decoding the image. In order to recapture the spatialinformation, a spectral-and-frequency-encoded (“SFE”) imaging techniquescan utilize a wavelength-dependent frequency modulation of theexcitation light before it is dispersed onto the sample via the grating.The fluorescence emission at each location can therefore be modulated inconcert with the frequency of the excitation light, thereby producing anadditional level of encoding.

Accordingly, it may be beneficial to address and/or overcome at leastsome of the deficiencies described herein above. For example, thereference interferometer signal could be used for active feedbackcontrol to correct non-linear movement of the scanning mirrors, therebyeliminating the need for post-acquisition processing.

OBJECTS AND SUMMARY OF THE INVENTION

One of the objectives of the present invention is to overcome certaindeficiencies and shortcomings of the prior art arrangements and methods(including those described herein above), and provide exemplaryembodiments of arrangements and methods for facilitatingphotoluminescence imaging, e.g., to obtain fluorescence images viaendoscopes, catheters, or small-diameter probes

According to certain exemplary embodiments of the present invention, thearrangements and methods for fluorescent imaging, e.g., spectrally andfrequency encoded (“SFE”) fluorescence imaging, can be provided, whichcan be performed in a sub-millimeter diameter endoscope with a highnumber of resolvable points.

A high number of resolvable points may be obtained within a smalldiameter probe, since the excitation bandwidth and the grating groovedensity govern the number of points in the image. For a given beamdiameter, the number of resolvable points attained by SFE is affected bythe excitation spectra of the fluorophore. Table 1 depicts the predictednumber of resolvable points for several common fluorescent labels,assuming beam diameters of 1.0 and 0.5 mm and a grating groove densityof 1500 lines/mm. For each case, the theoretical number of resolvablepoints either equals or exceeds that of fiber bundles of comparablediameter.

TABLE 1 Theoretical Number of Resolvable Points for Typical FluorophoresExcitation SFE # resolvable SFE # resolvable Fluorophore Bandwidth (nm)points (1.0 mm) points (0.5 mm) GFP 145 (375-520) 266,000 66,000 FITC 90 (430-520) 92,000 23,000 Cy5.5 110 (570-680) 89,000 22,000 ICG(plasma) 184 (670-854) 195,000 49,000 Table 1. Theoretical SFE number ofresolvable points for 1.0 mm and 0.5 mm beam diameters (rounded tonearest 4^(th) digit). SFE parameters: 1500 lines/mm grating, incidentillumination at Littrow's angle. Excitation bandwidth is defined as thefull width at 10% maximum.

Since exemplary SFE procedures may be conducted using a single opticalfiber, images obtained by these exemplary techniques may not containpixilation artifacts that are commonly observed in fiber bundles. (SeeE. Laemmel et al., “Fibered confocal fluorescence microscopy(Cell-viZio™) facilitates extended imaging in the field ofmicrocirculation—A comparison with intravital microscopy,” J. Vasc.Res., Vol. 41(5), 400 (2004)). Furthermore, flexibility of the SFEminiature probe will likely be greatly increased, as the bend radius fora single fiber is significantly less than that of imaging bundles. Theseexemplary advantages of SFE could be of significant benefit forapplications where image quality and maneuverability are of concern.

In addition, an exemplary spectral encoding technique according to anexemplary embodiment of the present invention can be utilized forfluorescence imaging using a swept source laser. In this exemplary casethe laser wavelengths can be rapidly tuned over the absorption band ofthe fluorophore. Each wavelength can be dispersed to a differentlocation on the sample. The collected sample fluorescence can then bedecoded as a function of time to reconstruct the image.

For example, according to one exemplary embodiment of the presentinvention, a reference interferometer signal can be used for activefeedback control to correct non-linear movement of the scanning mirrors,thereby eliminating the need for post-acquisition processing.

Indeed, according to one exemplary embodiment of the present invention,systems and methods can be provided for obtaining a photoluminescenceradiation from at least one portion of a sample. For example, using atleast one arrangement, it is possible to receive a first radiation anddisperse said first radiation into at least one second radiation and atleast one third radiation. The second and third radiations can beprovided to different locations of the portion. In addition, thephotoluminescence radiation can be received from the portion based onthe first, second, or third radiations.

Such arrangement can include a grating, a prism, a grism, a dualprism-grism and/or a lens. For confocal applications, the lens may havea numerical aperture that is greater 0.5. The arrangement may alsoinclude at least one optical fiber, which can have multiple claddings.The arrangement can include a plurality of optical fibers and/or atleast one of at least one pin hole arrangement or at least one slitarrangement. At least one of the optical fiber(s) can be a multimodefiber.

According to another exemplary embodiment of the present invention, awavelength tuning light source can be provided which may be configuredto provide the first radiation. Further, a light source can be includedand configured to provide the first radiation that has multiplewavelengths. In addition, a further arrangement can be provided whichmay be configured to modulate the wavelengths at different frequencies.The further arrangement can include an interferometric arrangement whichmay include at least one translatable component. The further arrangementmay include a further interferometric arrangement configured to correctfor non-linearities in the translatable component. A further arrangementmay include an acousto-optical, or electro-optical modulator to providethe frequency encoding.

In yet another exemplary embodiment of the present invention, thearrangement can be configured to generate information associated withthe different locations as a function of the photoluminescenceradiation. A processing arrangement can be provided configured togenerate at least one image based on the information. For example, theprocessing arrangement can be configured to receive the signal, andFourier transform the signal to generate the image. The image caninclude a microscopic image and/or an endoscopic image.

According to a further exemplary embodiment of the present invention,the arrangement can include a detecting arrangement which may beconfigured to receive the photoluminescence radiation and generate atleast one signal which can be associated with the photoluminescenceradiation. The arrangement can also be configured to be able to controla position of the second and third radiations on the different locationson the portion of the sample.

Other features and advantages of the present invention will becomeapparent upon reading the following detailed description of embodimentsof the invention, when taken in conjunction with the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Further objects, features and advantages of the present invention willbecome apparent from the following detailed description taken inconjunction with the accompanying figures showing illustrativeembodiments of the present invention, in which:

FIG. 1 is a block diagram of portions of a spectrally and frequencyencoded (“SFE”) system;

FIG. 2 is a block diagram of an exemplary embodiment of a Michelsoninterferometer arrangement, which can be used for spectral modulation,containing a stationary and scanning mirror in accordance with anexemplary embodiment of the present invention;

FIG. 3 is a schematic diagram of an exemplary embodiment of an apparatusaccording to the present invention which can be used to demonstrate anexemplary SFE procedure;

FIG. 4A is a first operational and schematic diagram providing detailson the operation of the exemplary embodiment of the SFE procedureaccording to the present invention;

FIG. 4B is a first operational and schematic diagram providing detailson the operation of the exemplary embodiment of the SFE procedureaccording to the present invention;

FIG. 5A is an exemplary SFE fluorescence image of microfluidic channelsfilled with Indocyanine Green (“ICG”—a fluorescent compound);

FIG. 5B is an exemplary reflectance image of microfluidic channelsfilled with ICG taken with an epi-illuminated microscope;

FIG. 6 is a detailed diagram of an exemplary SFE system utilizing asingle fiber probe in accordance with an exemplary embodiment of thepresent invention;

FIG. 7 is a detailed diagram of an exemplary embodiment of the SFE probeaccording to the present invention which uses and/or includes a singledual-clad fiber;

FIG. 8 is a detailed diagram of an exemplary embodiment of the SFE probeaccording to the present invention which uses a single-mode fiber forillumination and a large-core multimode fiber for collection offluorescence;

FIG. 9 is a detailed diagram of an exemplary embodiment of the SFE probeaccording to the present invention which uses multiple multimode fibersfor collection;

FIG. 10 is a detailed diagram of an exemplary embodiment of the SFEprobe according to the present invention which has another tip, where anorientation of lens and fibers have been modified;

FIG. 11 is a block diagram of an exemplary embodiment of a systemaccording to the present invention for correcting artifacts induced bynon-linear motions in an interferometer thereof;

FIG. 12 is a detailed diagram of an exemplary embodiment of the SFEprobe according to the present invention for microscopic applicationswhich uses a high numerical aperture lens and/or objective placed aftera grating;

FIG. 13 is a detailed diagram of an exemplary embodiment of the SFEprobe according to the present invention for microscopic applications inwhich an inline optic axis can be maintained via one or more prismsbefore and after the grating;

FIG. 14 is a detailed diagram of an exemplary embodiment of a confocalmicroscopic SFE probe according to the present invention which uses aslit and/or pinholes at a distal probe tip to increase a confocalsectioning;

FIG. 15 is a detailed diagram of a further exemplary embodiment of aconfocal microscopic SFE probe according to the present invention whichuses the slit and/or pinholes at a proximal end of collection fibers toincrease the confocal sectioning;

FIG. 16 is a set of graphs of results of exemplary spectral proceduresused to correct signals for non-linear motion of the scanning mirror inthe Michelson interferometers;

FIG. 17 is a flow diagram of an exemplary embodiment of the method forreconstructing the florescent image;

FIG. 18 is a flow diagram of an exemplary procedure used to obtain anexemplary excitation spectrum; and

FIG. 19 is a flow diagram of an exemplary embodiment of a spectralprocedure for correcting a non-linear mirror motion.

Throughout the figures, the same reference numerals and characters,unless otherwise stated, are used to denote like features, elements,components or portions of the illustrated embodiments. Moreover, whilethe subject invention will now be described in detail with reference tothe figures, it is done so in connection with the illustrativeembodiments. It is intended that changes and modifications can be madeto the described embodiments without departing from the true scope andspirit of the subject invention as defined by the appended claims.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

FIG. 1 shows a schematic diagram of -portions of a spectrally andfrequency encoded (“SFE”) system. For example, light 100 can be filteredby filter 105 to match the absorption band of the fluorophore, andprovided into an interferometer 110, which can be a Michelsoninterferometer, as well as a Sagnac, Mach-Zehnder, Twyman-Greeninterferometers, etc. The input light is affected by the interferometer110 so as to produce a spectral modulation on the input light. Light 130from the interferometer 110 then can illuminate a dispersive element 135which can precede or follow a lens. Each wavelength component with itsunique modulation frequency can be focused at a distinct location on afluorescent sample 140. Fluorescence in the sample 140 is excited, afluorescent light 145 returns through the grating lens pair 135, and canbe collected and directed to a detector 150.

The detected light can be processed via a Fourier transformation or thelike to recover a fluorescence intensity as a function ofone-dimensional location from the sample 140. Additional detectors maybe utilized to measure the excitation spectra and/or absorption and/ordiffuse reflectance spectra of the sample as a means for correcting forthe excitation spectrum shape and/or the absorption and/or scatteringartifacts in turbid samples. A second reference light 115, is directedto one component of the interferometer and utilized to compensate thenonlinearity of a moving component of the interferometer.

According to the exemplary embodiment illustrated in FIG. 1 anddiscussed above, broadband excitation light, which is possibly filteredto match the fluorophore's absorption spectrum, can be directed througha scanning Michelson interferometer. The scanning interferometerintroduced a wavelength-dependent frequency modulation whose intensitywas:

$\begin{matrix}{{{I_{ex}\left( {\lambda_{{ex},i},t} \right)} = {\frac{1}{2}{{I_{ex}\left( \lambda_{{ex},i} \right)}\left\lbrack {1 + {\cos\left( {\frac{4\pi}{\lambda_{{ex},i}}{vt}} \right)}} \right\rbrack}}},} & (2)\end{matrix}$where I_(ex)(λ_(ex,i)) is the spectral intensity corresponding to thei^(th) wavelength (λ_(ex,i)) of the excitation light, and v is thevelocity of the interferometer scanning mirror. As long as theinterferometer scans a large enough distance to provide sufficientspectral resolution, the number of resolvable points in SFE fluorescenceimaging is also governed by Eq. 1. After being dispersed and focusedonto the sample the modulated fluorescence light generates fluorescenceemission. Reconstruction of a single line in the SFE image is performedby taking the Fourier transform of the detected fluorescent signal,following correction by the reference interferometer signal. MultipleSFE lines are acquired as the probe is slowly scanned to create an SFEimage.Michelson Interferometer

A block diagram of an exemplary embodiment of an interferometer, e.g.,the Michelson interferometer, is shown in FIG. 2. As shown in FIG. 2,light 200 is incident on a beam splitter 215, which directs one portionof the light 200 to a stationary mirror 225 and the other portion of thelight 200 to a scanning mirror 230. The reflected beams return to thebeam splitter 215, and are combined to exit the interferometer asspectrally modulated light 235 which is incident upon the sample in thecase of the interferometer, or a detector for a reference measurement.The beam splitter 215 may be preceded by a filter 205 so as to filterout wavelengths not contained within the fluorophore's excitationspectrum. A compensator 220 may be inserted into one arm of theinterferometer to correct for a dispersion differences in the mirrorarms 225, 230.

Exemplary Embodiment of a Procedure According to the Present Invention

FIG. 3 depicts an exemplary embodiment of an apparatus according to thepresent invention which can be used to indicate the exemplary SFEprocedure, and FIG. 4 depicts an exemplary implementation of suchprocedure in more detail. For example, light from a broadband source 302can be delivered through a filter (F) 308 and single-mode fiber (SMF)310 to a Michelson interferometer containing a beam-splitter 312 and twomirrors (M), at least one of which may be scanned. The interferometermay generate a wavelength dependent frequency modulation on a broadbandlight 400. For example, a pair of a compact grating 316 a and a lens 316b as a pair 316 can be used to illuminate a sample 314, 420, thussimulating, e.g., a ˜1 mm diameter miniature endoscope.

The lens 316 b, 415 (e.g., f=12.5 mm) can focus each of uniquelymodulated excitation wavelengths 410 onto a different location of thesample 314 after they had been dispersed by the holographic transmissiongrating 316 a, 405 (being impacted by light 400 having 1200 lines/mm inFIG. 4), which can be any diffractive element. The fluorescence emission425 at each location can consequently be modulated at the frequencyf_(ex,i)=2v/λ_(ex,i) as shown in FIG. 4, where I_(em) is the entirefluorescence emission spectrum. Light from a Helium-Neon laser 306 wasalso directed into the interferometer through beam-pickoff 304 andsingle-mode fiber 310, and then deflected by beam-pickoff 318 through a633 nm bandpass filter (BPF) 320 for detection by an avalanchephotodiode (D_(R)) to obtain a reference signal for correction ofscanning mirror nonlinearities.

The emitted light was transmitted back through the same lens 316 b, 415and the grating 316 a, 405. The fluorescence 430 (also shown in FIG. 3as dashed lines), which can be diffracted at a different angle than thereflected light, can then be deflected by a mirror M shown in FIG. 3through an 830 nm long-pass filter LPF, and focused onto a secondavalanche photodiode D_(F) by a combination of spherical and cylindricallenses CL. In this exemplary configuration, the grating 316 a, 405 canbe mounted to a galvanometer, and rotated to provide the slow-axisscanning. A reconstruction of the exemplary image can be accomplished bytaking the Fourier-transform of the modulated fluorescence for eachgalvanometer scan angle after using the reference signal to correct theinterferogram.

Indocyanine green (“ICG”) can be used, which is a near-infraredfluorophore (e.g., having an excitation 650-850 nm, and an emission805-950 nm) that has been FDA approved for several clinical indications(see C. H. Tung, “Fluorescent peptide probes for in vivo diagnosticimaging,” Biopolymers, Vol. 76(5), 391, (2004)), and modified fortargeted antibody labeling (see S. Ito et al., “Detection of humangastric cancer in resected specimens using a novel infrared fluorescentanti-human carcinoembryonic antigen antibody with an infraredfluorescence endoscope in vitro,” Endoscopy, Vol. 33(10), 849, (2001),and T. Bando et al., “Basic studies on a labeled anti-mucin antibodydetectable by infrared-fluorescence endoscopy,” Journal ofGastroenterology, Vol. 37(4), 260 (2002)).

FIG. 5A shows a fluorescence image 500 of microfluidic channels (asdescribed in M. Shin et al., “Endothelialized networks with a vasculargeometry in microfabricated poly(dimethyl siloxane),” BiomedicalMicrodevices, Vol. 6(4), 269 (2004)) filled with ICG (2 mg ICG/mL DMSO)obtained using the SFE technique. A corresponding epi-illuminatedreflectance micrograph image 505 is shown in FIG. 5B which demarcatesthe smallest, 35 μm wide channels. The exemplary SFE image can becollected at 2 Hz with a 1.4 mm×1.4 mm field of view. The centerwavelength and spectral range (full-width at 15% maximum) ofillumination after low-pass filtering can be 780 nm and 50 nm,respectively. The Michelson interferometer scanning mirror can betranslated ±0.25 mm around the zero-path-length difference position toprovide a spectral resolution of ˜0.6 nm, corresponding to a theoretical83 resolvable points. (See J. Kauppinen et al., Fourier Transforms inSpectroscopy, Wiley-VCH, New York, p. 271, (2001)). The probe input beamcan be 1.1 mm in diameter, and may be incident on the grating at 22°,also likely resulting in a spectral resolution of 0.6 nm. Since theusable excitation bandwidth of ICG can be 125 nm in DMSO, this probeconfiguration could theoretically enable >200 resolvable points perfrequency-encoded line. However, the limited bandwidth of the currentsource can reduce this number to, e.g., 84.

Exemplary lateral resolution measurements along the spectrally encodedline can be estimated by measuring the edge response function of, e.g.,15 lines in the image along the wavelength-encoded axis at verticaledges in the microfluidic channels. The exemplary measurements candemonstrate a spatial resolution of 15.9±4.9 μm (mean±stdev),corresponding to a total of approximately 88 resolvable points acrossthe field of view. This is in approximate agreement with the expectedcalculations. The resolution along the transverse axis can be limited bythe imaging optics.

The total number of resolvable points obtained by the exemplaryembodiment of the SFE fluorescence imaging apparatus according to thepresent invention, which approximates the dimensions of a 1 mmendoscope, can be, e.g., n² =7,744. This exemplary value can becomparable to state-of-the-art fiber-bundle based technologies ofsimilar diameter, and may be improved by increasing the excitationbandwidth or utilizing a higher density grating while simultaneouslyincreasing the scanning range of the interferometer.

Fiber-optic Exemplary Embodiment: Single Dual-clad Fiber

Exemplary SFE techniques can be advantageous in that high qualityimaging may be obtained using a single optical fiber. In order tominimize size in the development of future SFE endoscopes, it may beadvantageous to collect the fluorescent emission through the probegrating. Stokes-shifted fluorescent light, however, may not couple backto the core of a single-mode illumination fiber. An exemplary solutionto this challenge may be the use of a dual-clad fiber (as described inD. Yelin et al., “Double-clad fiber for endoscopy, ” Opt. Lett., Vol.29(20), 2408 (2004)) such that excitation light can be transmittedthrough a central core, and the fluorescence may be obtained through theinner cladding. Using exemplary ray-tracing models, this exemplaryapproach is effective without significantly increasing the probediameter or compromising resolution.

FIG. 6 shows a detailed diagram of an exemplary SFE system utilizing asingle dual clad fiber probe in accordance with an exemplary embodimentof the present invention for collecting fluorescent images. For example,illumination light generated by a source 600 can be filtered by a filter605. Such filtered light, and light from a reference 625 can be directedthrough a Michelson interferometer including a beam splitter 610, astationary mirror 615, and a moving mirror 620. The light provided fromthe reference 625 can be deflected by a notch filter or a reference beamsplitter 622 to a reference detector 630. The illumination light canpass through the reference beam splitter 622 and a dichroic filter orbeam splitter 635, and can be coupled via a coupler to a centralsingle-mode core of a dual clad fiber 645. Light 655 exiting the core ofthe fiber 645 can be dispersed and focused onto a sample 660 by adispersive element and lens 650. The emitted fluorescence can returnthrough a lens 650 and the dispersive element 650. Since such light isStokes shifted, it may not easily couple back into the single-mode core,but would likely couple to the inner cladding of the dual-clad fiber645. The emitted fluorescence emerging from the proximal end of thefiber can be deflected by the dichroic filter or beam splitter 635 to afluorescence detector 640. On the other hand, the reflected light cancouple directly back into the single-mode core. After passing throughthe dichroic filter or beam splitter 635, this signal can be directed toan additional detector for a reconstruction of a spectrally encodedreflectance image.

FIG. 7 shows an exemplary embodiment of a distal end of a probeaccording to the present invention which is configured to utilize theexemplary apparatus shown in FIG. 6 and using a dual clad-fiber. Forexample, spectrally modulated light 700 from the interferometer can beprovided to a central core 715 of a dual-clad fiber 710 via couplingoptics 705. Illumination light 720 emerging from the core 715 can bedivergent, and collimated by a lens 725, which can be a micro-lens, GRINlens, etc. Collimated light 730 can be dispersed by a dispersive element735, which may be a transmissive grating, a reflective grating, prism,hologram, or any other diffractive element. Dispersed light 745 can befocused onto a sample 750 by a lens 740, thus causing a fluorescenceemission. The spectrally modulated emitted fluorescence 755 can begathered by and collimated by the lens 740, transmitted through agrating 735, and focused by the lens 725 to an inner cladding 760 of thedual-clad fiber 710. The modulated fluorescence 755 can then betransmitted back down the inner cladding 760 for detection.

Multiple Fiber Exemplary Embodiments: Two or More Fibers

The exemplary SFE arrangements can also be configured to be provided ina multiple fiber configuration. In such exemplary embodiment of thepresent invention, a single-mode fiber can be used to send theillumination light to the sample, and one or multiple multi-mode fibersmay be used to collect the emitted fluorescence. The reflectance imagecan be reconstructed because the reflected light would couple back tothe illumination fiber.

FIG. 8 an exemplary embodiment of a distal end of a probe according tothe present invention which is configured to utilize the exemplaryapparatus shown in FIG. 6 and using two optical fibers. The descriptionof this exemplary arrangement is similar to that of FIG. 7 providedherein, and elements thereof have the same description, except thatelements 700, 705, etc. have been replaced with elements 800, 805, etc.,respectively, except as indicated below. In the exemplary embodiment ofFIG. 8, the spectrally modulated illumination light can be transmittedto the distal end of the probe via single-mode fiber 815. The emittedfluorescence can be coupled back to the core of a multimode fiber 860.In another exemplary embodiment of the present invention shown in FIG.9, a multimode core 860 of FIG. 8 can be replaced by a linear array ofmultimode fibers 960.

Alternate Exemplary Probe Tip Embodiment

Another exemplary embodiment of a distal end of the probe according tothe present invention is shown in FIG. 10. Although an exemplarymultiple-fiber embodiment is shown in FIG. 10, this exemplary embodimentof the probe tip can be used in all fiber configurations. In thisexemplary embodiment, the ordering of the optics can be changed. Forexample, illumination light 1020 diverging from the illumination fibercan be passed through a lens 1025, which can be a GRIN lens, and/orother types of lenses or objectives. The fiber-lens separation can beselected such that the light begins to converge to a focus beyond agrating 1035 and on a sample 1050. The emitted fluorescence can thenpass through the grating, and may be focused onto collection fiber(s) bythe lens 1025.

Fluorescence Microscopy and Fluorescence Confocal Microscopy

Similar to exemplary spectrally encoded confocal imaging procedures, theexemplary SFE procedures can also be implemented in configurations thatmay enable endoscopic fluorescence microscopy. For these exemplaryapplications, it may be advantageous to utilize an imaging lens with ahigh magnification or numerical aperture. For certain exemplaryendoscopic microscopy configurations, a numerical aperture can begreater than 0.3, and may preferably be greater than 0.5. Due toaberrations that may occur when large angles illuminate the grating, asshown in FIG. 12, it may furthermore be advantageous to place a grating1225 prior to a lens 1230 as is conducted in a technique termedspectrally-encoded confocal microscopy (SECM). (See G. J. Tearney etal., “Spectrally encoded confocal microscopy, ” Opt. Lett., Vol. 23(15),1152 (1998); and C. Pitris et al., “A GRISM-based probe for spectrallyencoded confocal microscopy, ” Optics Express, Vol. 11(2), 120 (2003)).

FIG. 13 shows a further exemplary embodiment of the arrangementaccording to the present invention which can utilize one or more prisms1325, 1335 in front or behind a grating 1330 in order keep spectrallydispersed light 1345 along the same axis as the optical probe. Similarto macroscopic imaging, exemplary multiple core/fiber combinations maybe utilized to collect the fluorescent light. For implementing confocalfluorescence microscopy procedures in which the optical sectioning depthcan be defined by an axial response function, as shown in FIG. 14, anadditional spatial filter comprising one or more fiber apertures 1465(or a physical aperture such as a slit or pinhole 1460) may be placed atthe distal fiber tip of the collection fiber(s) to reject out-of-focuslight. Alternatively, as shown in FIG. 15, such exemplary spatial filter1570 may be placed at the proximal end of the detection fibers in afiber bundle or array of fibers.

Exemplary Reference Interferometer

Even slight (<1%) non-linearities in the translation of the scanningmirror of the Michelson interferometers can cause incorrect spectralinformation, both in line-shape and frequency (wavelength), thereforedistorting or ruining the image. This can be corrected as using theexemplary embodiment of the arrangement according to the presentinvention which is depicted as a block diagram in FIG. 11. For example,as shown in FIG. 11, normal or broadband illumination light 1100 canpass through a Michelson interferometer 1105, and may emerge asspectrally modulated light 1110. This modulated light 1110 can betransmitted to a fluorescent sample or a reference illumination detector1115. Single-frequency light 1120 (e.g., a laser, such as a Helium-Neonlaser, etc., or provided from another source, etc.) may also passthrough the Michelson interferometer 1105. A resultant spectrallymodulated light 1125 can be detected by a reference correction detector1130. The spectrally modulated light 1125 can have a single modulationfrequency with equally spaced zero-crossings. However, non-linearitiesin the motion of the scanning mirror can change the spacing of thezero-crossings, and may result in an incorrect reconstruction of spectraand image lines when the Fourier transform is performed.

Exemplary embodiments of a correction procedure according to the presentinvention as described below can result in a re-interpolation of thedata signals, based on the a priori knowledge that the reference signalshould have equally-spaced zero-crossings. FIG. 16 shows exemplarygraphs associated with representative data with respect to this point.For example, the original reference signal detected by the referencecorrection detector 1130 is illustrated in a first graph 1600. Thecorrection factor of the upper middle panel can be used tore-interpolate the original data to obtain the trace in a second graph1610. A further graph 1605 illustrates a Hilbert transform of areference signal used to correct the data. The Fourier transform of theuncorrected data is shown in a fourth graph 1615 (e.g., spectrum ofmonochromatic reference signal prior to correction, obtained as aFourier transform of the first graph 1600) and a spectrum ofmonochromatic reference signal after correction, obtained as a Fouriertransform of the second graph 1610 is shown in a fourth graph 1620. Thegraphs 1615 and 1620 demonstrate the recovery of a single-frequencysignal when the correction factor is applied to the reference dataitself. Exemplary applications to other detected signals can be equallyeffective in a spectral correction procedure.

Exemplary Excitation Spectra Measurement

Because the illumination light can be dispersed onto the sample toprovide spectral encoding, each spot on the sample can be illuminatedwith a different wavelength. For example, by scanning the spectrallyencoded line along the sample, approximately parallel to the line ofdispersion, each point can be sequentially illuminated by the fullbandwidth of the illumination light. By monitoring the intensity at eachpoint as the wavelengths are scanned, the excitation spectrum can berecovered for each location on the sample.

Emission Spectra Measurement

An exemplary embodiment of the SFE procedure according to the presentinvention can allow for a recovery of the emission spectrum. Each pointon the sample can be illuminated by a different wavelength, each ofwhich may be encoded with a different modulation frequency. For example,if some or all of the emitted light is coupled into a spectrometer, theemission spectrum can be recovered by conventional procedures and/ormethods. The spectrometer can be dispersive and/or Fourier transformtype. The Fourier transform-type spectrometer may be a secondinterferometer added to the exemplary system. By scanning the spectrallyencoded line in both directions (e.g., one to form the image, and theother to collect the excitation spectra), the excitation-emission matrixcan be reconstructed for each point in the image.

Lifetime Measurement

According to further exemplary embodiments of the present invention, itis also possible to determine the fluorescence lifetime at each locationin the image. As indicated in Eq. 2, the illumination light oscillatessinusoidally, forcing the fluorescence emission to oscillate in the samemanner. However, the fluorescence may be emitted with a slight phaseshift (φ) and a decreased amplitude:

$\begin{matrix}{{{I_{{em},j}\left( {\lambda_{{ex},i},\lambda_{{em},j},t} \right)} = {{m\left( {\lambda_{{ex},i},\lambda_{{em},j}} \right)}\frac{1}{2}{{I_{ex}\left( \lambda_{{ex},i} \right)}\left\lbrack {1 + {\cos\left( {{\frac{4\pi}{\lambda_{{ex},i}}{vt}} - {\varphi\left( {\lambda_{{ex},i},\lambda_{{em},j}} \right)}} \right)}} \right\rbrack}}},} & (3)\end{matrix}$where m is the demodulation factor which depends on both the excitationand emission wavelengths. The fluorescence lifetime can be measured as:

$\begin{matrix}{{{\tau\left( {\lambda_{ex},\lambda_{em}} \right)} = \frac{\tan\left\lbrack {\varphi\left( {\lambda_{ex},\lambda_{em}} \right)} \right\rbrack}{2\pi\;{f\left( \lambda_{ex} \right)}}},} & (4)\end{matrix}$where φ(λ_(ex),λ_(em)) is the phase difference between the illuminationlight and the emitted fluorescence. Lifetime can also be calculated as

$\begin{matrix}{\tau = {\frac{\sqrt{1 - \left\lbrack {m\left( {\lambda_{ex},\lambda_{em}} \right)} \right\rbrack^{2}}}{2\pi\;{f\left( \lambda_{ex} \right)}{m\left( {\lambda_{ex},\lambda_{em}} \right)}}.}} & (5)\end{matrix}$Exemplary Light Sources

In order to attain a high number of resolvable points shown in Table 1,it is preferable for the source to be capable of illuminating the entireexcitation spectrum. This can be made possible through the use of, e.g.,thermal lamps, arc lamps, solid-state lasers, and LEDs. Additionally,alternative sources such as supercontinuum generation with photoniccrystal fiber technology can be utilized, the description of which isprovided in G. McConnell, “Confocal laser scanning fluorescencemicroscopy with a visible continuum source, ” Opt. Express, Vol. 12(13),2844 (2004). The use of broadband NIR lasers as a light source canfacilitate SFE two-photon fluorescence imaging.

Alternatively, the fluorescence imaging can be accomplished withswept-source lasers. In this case the frequency encoding is notnecessary and the technique reduces to traditional spectral encodingbecause the individual locations of the sample are sequentiallyilluminated. It is still possible to obtain excitation, emission, andlifetime spectra with this embodiment, as well as reconstruction of theEEM.

Exemplary Procedures for Reconstructing a Fluorescent Image

An exemplary embodiment of the method according to the present inventionfor reconstructing the fluorescent image begins with the flow diagram ofFIG. 17. As shown in this figure, Eq. 2 is inverse Fourier transformedin step 1710. The resultant signal may likely be one line in the image.Then, intensity corrections can be accomplished by dividing by theillumination spectrum, as indicated in step 1720.

An exemplary excitation spectrum can be obtained using the exemplaryprocedure of FIG. 18, in which, after scanning along the direction ofthe spectrally encoded line, Eq. 3. is inverse Fourier transformed instep 1810. Then, in step 1820, source cross-correlation spectra isdivided (e.g., by an inverse Fourier transform of Eq. 2). An amplitudeof resultant signal is an exemplary excitation spectra, and the phase ofresultant signal can be related to time constants via Eq. 4, whereφ(λ_(em),λ_(ex)) may be the phase difference between the and the sourcecross-correlation (φ_(I)(λ_(ex))).

The exemplary excitation lifetime can also be obtained. For example, theinverse Fourier transform of Eq. 3 for a phase of the resultant signalcan be the phase of the fluorescence φ(λ_(em),λ_(ex))) The inverseFourier transform of Eq. 1 for the phase of the resultant signal can bethe phase of the source cross-correlation (φ_(I)(k_(X))). The phasedifference may be related to the fluorescence lifetime via Eq. 4 where:φ(λ_(em),λ_(ex))=φ(λ_(em),λ_(ex))−φ_(I)(λ_(ex)).  (6)

An exemplary embodiment of a spectral procedure for correctingnon-linear mirror motion according to the present invention is shown ina flow diagram of FIG. 19. Each full scan (e.g., back and forth) of themirror can correspond to two lines in the image. This exemplarycorrection procedure can utilize a simultaneous acquisition of twointerferograms for each line in the image, e.g., (a) the signal ofinterest, and (b) a signal with a known spectrum, preferably asingle-frequency source such as a He—Ne laser, which can be used as areference to correct the time-traces for non-linear motion of thescanning mirror of the Michelson interferometer.

As shown in FIG. 19, for example, data is truncated to linear region ofscan, containing 2^(n) data points for efficient Fast Fourier transforms(usually symmetric about the center point, dictated by duty cycle of thedriving waveform) in step 1910. In step 1920 the mean of the data issubtracted from each signal if acquisition was not AC-coupled (e.g., seethe first graph 1600 of FIG. 16 showing only a small portion of data).The signals are then time-reversed for most or all odd scans (e.g., tocorrect for the opposite direction of the scan) in step 1930. In step1940, the Hilbert transform of the reference signal is taken. Forexample, the Fourier transform of a single-frequency laser (e.g., thereference) can be an infinite sine wave with equally spacedzero-crossings. Further, the Hilbert transform can be based on the FastFourier transform, and the imaginary part may correspond to the originaldata with a 90° phase shift.

Then, in step 1950, the unwrapped phase of the Hilbert transform of thereference signal is taken (as shown in the graph 1605 of FIG. 16). Thiscan correspond to the actual position of the mirror as a function oftime, and may be monotonically increasing (e.g., not locally linear).Then, a new linear mirror position can be generated ranging from theminimum to maximum of the actual mirror position—step 1960. In step1970, the signal of interest can be re-interpolated onto the new linearmirror position space, e.g., using the unwrapped phase of the referenceHilbert transform. The second graph 1610 of FIG. 16 shows the signal ofinterest which is the reference itself. The spectrum of interest canthen be determined from the Fourier transform of the interpolated signalin step 1980. For example, see the third and fourth graphs 1615 and 1620for uncorrected and corrected signals, respectively.

Alternatively, the time-trace of reference signal can be used togenerate a clock signal which may be used to gate the data acquisitionat equally spaced mirror locations, thereby automatically correcting thedata, and obfuscating the post-processing algorithm described above.

Exemplary Components

Various components can be used for the exemplary embodiments of thepresent invention. Provided below are merely samples of such components,and in no way limit the scope of the present invention.

For example, the broad bandwidth light source can include LED, filamentlamp (e.g. Tungsten-halogen, Mercury, Xenon, Deuterium), array of diodelasers, continuum generation source, femtosecond solid-state source,semiconductor optical amplifier, rare-earth doped fiber, ASE source, dyefluorescence, SLED, swept-source laser, etc. The reference source caninclude a monochromatic light source, such as HeNe laser, gas laser,diode laser, filtered broad bandwidth light source, etc. The opticalfiber can include Dual-clad fiber, single-mode fiber, multimodefiber(s), photonic crystal fiber, hollow-core fiber, hollow waveguide,etc.

Further, the dispersive element can include transmission grating,reflection grating, hologram, prism, etc. The compensator can include aneutral density filter. The dispersion compensator can include dualopposing prisms or optical glass, crystal or other dispersion modifier,etc. The wavelength dependent frequency can be one or more of thefollowing: Scanning mirror via galvanometer, piezoelectric transducer,or solenoid. Rapidly scanning optical delay line (RSOD) as described inG. J. Tearney et al, “High-speed phase- and group-delay scanning with agrating-based phase control delay line,” Optics Letters, Vol. 22(23),1811 (1997), phase control delay line, acousto-optic modulator,electro-optic modulator, spinning helical cam, rotating hologram,spinning mirror array, spinning cube, piezoelectric fiber stretcher,variable reflectance plate beam splitter (Fabry-Perot interferometer),etc.

The exemplary interferometer can include any arrangement for combininglight returned from two arms, such as, e.g., Mach-Zehnder, Sagnac,Michelson, Fabry-Perot interferometers. It is noted that the reflectionfrom these arms is not necessary, and such arrangements can operate in atransmission mode. It is also possible to incorporate polarization beamsplitters, common path elements and/or circulators in such exemplaryarrangements. The dichroic splitter can include an interference filter,diffraction grating, dichroic mirror, etc. The exemplary spectraldispersion can be accomplished using Grating spectrometer, FourierTransform spectrometer, prism spectrometer, etc. The exemplary detectorscan include photodiode, photomultiplier tube, avalanche photodiode, CCD,etc.

The foregoing merely illustrates the principles of the invention.Various modifications and alterations to the described embodiments willbe apparent to those skilled in the art in view of the teachings herein.Indeed, the arrangements, systems and methods according to the exemplaryembodiments of the present invention can be used with any OCT system,OFDI system, SD-OCT system or other imaging systems, and for examplewith those described in International Patent ApplicationPCT/US2004/029148, filed Sep. 8, 2004, U.S. patent application Ser. No.11/266,779, filed Nov. 2, 2005, and U.S. patent application Ser. No.10/501,276, filed Jul. 9, 2004, the disclosures of which areincorporated by reference herein in their entireties. It will thus beappreciated that those skilled in the art will be able to devisenumerous systems, arrangements and methods which, although notexplicitly shown or described herein, embody the principles of theinvention and are thus within the spirit and scope of the presentinvention. In addition, to the extent that the prior art knowledge hasnot been explicitly incorporated by reference herein above, it isexplicitly being incorporated herein in its entirety. All publicationsreferenced herein above are incorporated herein by reference in theirentireties.

1. A system for obtaining a photoluminescence radiation from at leastone portion of a sample, comprising: at least one arrangement configuredto: i. receive a first radiation and spatially and spectrally dispersethe first radiation into at least one second radiation and at least onethird radiation, the second and third radiations being provided todifferent locations of the at least one portion, and ii. receive thephotoluminescence radiation from the at least one portion based on thesecond and third radiations, wherein at least one of the second andthird radiations excites at least one portion of the at least one sampleto generate the photoluminescence radiation.
 2. The system according toclaim 1, wherein the at least one arrangement comprises at least one ofa grating, a prism, a grism, a dual prism-grism or a lens.
 3. The systemaccording to claim 1, wherein the at least one arrangement comprises alens having a numerical aperture that is greater 0.5.
 4. The systemaccording to claim 1, wherein at least one first arrangement comprisesat least one optical fiber.
 5. The system according to claim 4, whereinthe at least one optical fiber has multiple claddings.
 6. The systemaccording to claim 4, wherein the at least one optical fiber includes aplurality of optical fibers.
 7. The system according to claim 4, whereinthe at least one arrangement comprise at least one of at least one pinhole arrangement or at least one slit arrangement.
 8. The systemaccording to claim 4, wherein at least one of the at least one opticalfiber is a multimode fiber.
 9. The system according to claim 1, furthercomprising a wavelength tuning light source configured to provide the atleast one first radiation.
 10. The system according to claim 1, furthercomprising a light source configured to provide the at least one firstradiation that has multiple wavelengths.
 11. The system according toclaim 1, further comprising a further arrangement configured to modulatewavelengths of at least one of the second radiation or the thirdradiation at different frequencies.
 12. The system according to claim11, wherein the further arrangement comprises an interferometricarrangement.
 13. The system according to claim 12, wherein theinterferometric arrangement includes at least one translatablecomponent.
 14. The system according to claim 13, wherein the furtherarrangement comprises a further interferometric arrangement configuredto correct for non-linearities in the at least one translatablecomponent.
 15. The system according to claim 11, wherein the furtherarrangement includes at least one of an acousto-optical modulator or anelectro-optical modulator configured to provide frequency encodingcapabilities.
 16. The system according to claim 1, wherein the at leastone arrangement is configured to generate information associated withthe different locations as a function of the photoluminescenceradiation, and further comprising a processing arrangement configured togenerate at least one image based on the information.
 17. The systemaccording to claim 16, wherein the processing arrangement is configuredto receive the at least one signal, and to Fourier transform the atleast one signal to generate the image.
 18. The system according claim16, wherein the at least one image includes at least one of amicroscopic image or an endoscopic image.
 19. The system according toclaim 14, wherein the at least one arrangement comprises a detectingarrangement which is configured to receive the photoluminescenceradiation and generate at least one signal which is associated with thephotoluminescence radiation.
 20. The system according to claims 1,wherein the at least one arrangement is configured to be able to controla position of the second and third radiations on the different locationson the at least one portion of the sample.
 21. A method for obtaining aphotoluminescence radiation from at least one portion of a sample,comprising: receiving a first radiation and spatially and spectrallydispersing the first radiation into at least one second radiation and atleast one third radiation, the second and third radiations beingprovided to different locations of the at least one portion, andreceiving the photoluminescence radiation from the at least one portionbased on the second and third radiations, wherein at least one of thesecond and third radiations excites at least one portion of the at leastone sample to generate the photoluminescence radiation.
 22. The methodaccording to claim 21, further comprising: generating informationassociated with the different locations as a function of thephotoluminescence radiation; and generating at least one image based onthe information.
 23. A system for obtaining a photoluminescenceradiation from at least one portion of a sample, comprising: at leastone arrangement configured to: i. receive a first radiation andspatially and spectrally disperse the first radiation into at least onesecond radiation and at least one third radiation, the second and thirdradiations being provided to different locations of the at least oneportion, ii. receive the photoluminescence radiation from the at leastone portion based on the second and third radiations, and iii. modulatewavelengths of at least one of the second radiation or the thirdradiation at different frequencies.
 24. A method for obtaining aphotoluminescence radiation from at least one portion of a sample,comprising: receiving a first radiation and spatially and spectrallydispersing the first radiation into at least one second radiation and atleast one third radiation, the second and third radiations beingprovided to different locations of the at least one portion, receivingthe photoluminescence radiation from the at least one portion based onthe second and third radiations; and modulating wavelengths of at leastone of the second radiation or the third radiation at differentfrequencies.